Real-time quantitative RT-PCR (qRT-PCR) is the most sensitive technique for mRNA detection and quantitation currently available. qRT-PCR is also the preferred method for validating array analysis results, RNA interference experiments, and other techniques that evaluate gene expression changes. Ambion's Cells-to-Signal™ Kit (patent pending) is an improvement to the popular Cells-to-cDNA™ II Kit for cell lysate preparation for direct use in real-time qRT-PCR experiments. The kit uses a chemical lysis method to create cell lysates in less than 5 minutes at room temperature. The cell lysate can be used directly for RT-PCR when PCR primers are designed to span intron-exon boundaries, bypassing RNA isolation altogether. Lysates can be stored for future experiments. The Cells-to-Signal procedure is highly reproducible and is compatible with a wide variety of cell types. These improvements result in a single-step procedure that is optimized for high throughput analyses -- both for manual processing and on robotic platforms.

AM2050,AM8728

Compatible with TaqMan Gene Expression Assays

Applied Biosystems® pre-designed primer and probe sets for real-time PCR are one of the most commonly used methods for measuring changes in gene expression by qRT-PCR. The Cells-to-Signal technology is compatible with TaqMan Gene Expression Assays (formerly known as Assays-on-Demand™). Figures 1 and 2 show data generated in one-step real-time RT-PCR using TaqMan Gene Expression Assays for human GSK-3ß and VEG-F. In addition, the data (Figure 2) also demonstrate that the Cells-to-Signal procedure is sensitive enough to detect target in lysate equivalent to 3 cells.

Figure 1. qRT-PCR Detection of GSK-3ß Using TaqMan® Gene Expression Assays and Cells-to-Signal™. HeLa cells were lysed at concentrations of 1000, 200, 40, 8, and 1.6 cells/µl according to the Cells-to-Signal lysis procedure. 6 µl of cell lysate was used in a 20 µl one-step RT-PCR using a TaqMan Gene Expression Assay primer and probe set specific for GSK-3ß.

Figure 2. qRT-PCR Detection of VEG-F Using TaqMan® Gene Expression Assays and Cells-to-Signal™. HeLa cells were lysed at concentrations of 1000, 100, 10 and 1 cells/µl according to the Cells-to-Signal lysis procedure. 3 µl of cell lysate was used in a 10 µl one step RT-PCR using a TaqMan Gene Expression Assay primer and probe set specific for VEG-F.

Measuring siRNA Induced Knockdown

siRNA induced knockdown of gene expression is now one of the most widely used methods to determine gene function. Scientists at Ambion performed a study using cultured HeLa and MCF-7 cells transfected with 3 nM of chemically synthesized siRNA targeting GAPDH to demonstrate the effectiveness of the Cells-to-Signal Kit for measuring knockdown of gene expression (Figure 3). As expected, the expression levels of both GAPDH was down-regulated by more than 80% compared to cells transfected with a scrambled control, while the expression of 18S rRNA was unchanged.

Figure 3. Cells-to-Signal™ is Ideal for Measuring siRNA Induced Knockdown of Gene Expression. HeLa cells were plated in a 24 well plate at 30,000 cells/well. Cells were transfected with either a scrambled control (green) or with an siRNA to GAPDH (blue) at a concentration of 30 nM. After 48 hours, the cells were harvested and lysed in the Cells-to-Signal Lysis Buffer in a final volume of 500 µl. 3 µl of the cell lysate was used in one step RT-PCR, using a TaqMan® primer and probe set for GAPDH. GAPDH signals were normalized using 18S as an internal control (pink).

Ideal for High Throughput

The quick and simple Cells-to-Signal protocol can be easily adapted to a 96 or 384 well format, thus enabling the simultaneous analyses of multiple cell samples. The protocol will afford excellent reproducibility as seen in Figure 4 where VEG-F and GAPDH expression levels were determined in HeLa and MCF-7 cells grown in a 96 well plate.

Figure 4. Measurement of GAPDH and VEG-F Expression Levels in a 96 well Format Using Cells-to-Signal™. 3000 HeLa or MCF-7 cells were plated in 48 wells each of the same 96 well plate. Cells were grown for 24 hours, media was removed, and cells were washed once with 200 µl of 1X PBS. 200 µl of the Cells-to-Signal Lysis Buffer were added to the cells, and the plates were incubated for 5 min at room temperature. 3 µl of lysate were used in a 10 µl one-step RT-PCR using TaqMan® Gene Expression Assay for GAPDH or VEG-F. The two amplification curves on the left are for GAPDH, and the curves on the right are for VEG-F. The amplification curves in red and dark green were generated using HeLa cell lysate. The amplification curves in purple and light green were generated using lysate prepared from MCF-7 cells.

SYBR® Green Detection

Real-time detection of PCR products using SYBR Green is a commonly used method to quantitate gene expression by RT-PCR. Though SYBR Green detection requires more optimization than other real-time PCR detection techniques, it is economical and can be carried out on virtually any real-time detection platform. The Cells-to-Signal Kit is compatible with SYBR Green detection used with two-step RT-PCR.

Long-term Stability

When working with multiple cell samples, it is not always feasible to simultaneously process them all. Likewise, cell lysates may be needed at a later stage for repeating prior experiments or for quantitating the expression of different genes from the same sample. Lysates made with the Cells-to-Signal Kit can be safely stored at -20°C for two months or longer without RNA degradation (Figure 5).

Figure 5. Stability of Cells-to-Signal™ Lysates. HeLa cells were lysed at a concentration of 1000 cells/µl using the Cells-to-Signal Lysis Buffer. 100 µl aliquots of the lysate were stored for two months at -80ºC or -20ºC. Ambion's RNAqueous® Kit was then used to isolate total RNA from each aliquot along with an aliquot of lysate that was freshly prepared (not stored). RNA isolated from an equivalent number of cells processed using the RNAqueous procedure is shown on the far right. The RNA samples were analyzed on an RNA LabChip® using the Agilent 2100 bioanalyzer and show rRNA bands

Complete Kit for Cell Lysis and cDNA Synthesis

The Cells-to-Signal Kits contain reagents for 30 or 100 reverse transcription reactions, including M-MLV RT, oligo(dT)18 primers, and random decamer primers. Up to 5 x 10
4 cells per reaction can be used for the lysis step; up to 30% of the RT reaction volume can consist of cell lysate. Ambion scientists have used the Cells-to-Signal Kit with cell lines including HeLa, HeLa S3, 293, A549, MCF-7, K562, SKNAS and NHDF-neo cells, and we continue to test other cell types. The kit contains a positive control RNA and complementary primers for optimizing the procedure. This control can be added with the lysis solution to monitor the efficiency of the procedure. SuperTaq™ Polymerase is available separately.